Name: investigation of spatial interaction between astrocytes and neurons in cleared brains
Uploaded: 2022-03-31T14:50:00
Description: Watch this Scientific Journal Video about Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains at JoVE.com

该项目已获得欧洲研究理事会（ERC）根据欧盟地平线2020研究和创新计划（赠款协议No 803589），以色列科学基金会（ISF赠款No.1815/18）和加拿大 - 以色列赠款（CIHR-ISF，第2591/18号赠款）的资助。我们感谢Nechama Novick对整个手稿的评论。

实验方案由希伯来大学动物护理和使用委员会批准，并符合国家卫生研究所实验室动物护理和使用指南的指导方针。
1. 病毒载体转导
注意：病毒载体转导用于表达大脑中的荧光团。
<ol><li>使用图集（例如，Allen Brain Atlas）来定位目标区域的相关协调。 注意：3D地图集可以在线找到（例如，http://connectivity.brain-map.org/3d-viewer）。</li>
	<li>使用...

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组织清除方法是大脑研究中的革命性工具，引发了以前无法提出的问题。通过靶向一小群细胞，单个细胞甚至单个突触的特性，CLARITY现在可以通过使用相关的荧光团来靶向总细胞群或远距离连接特征。
荧光基团表达和CLARITY程序组合的结果不是二元的;许多因素可能会干扰导致次优结果的程序。首先，荧光基团的表达必须事先验证。由于CLARITY程序会导致一些信息丢失，因此在C...

近年来，星形胶质细胞功能以及它们如何与神经元回路相互作用的知识急剧增加。星形胶质细胞可以影响可塑性1，2，帮助神经元损伤后恢复3，4，甚至诱导 从头 神经元增强，最近的研究表明星形胶质细胞在记忆获取和奖励中的重要性，以前被认为是纯粹的神经元功能5，6，7.星形胶质细胞研究中特别感兴趣的一个特征是细胞的空间排列，其在海马体和其他大脑结构中保持独特的空间组织8，9，10。与在细胞体瘤之间交织的神经元树突不同，海马星形胶质细胞栖息在视觉上可区分的区域，其过程之间略有重叠，形成不同的结构域8，11，12，13。支持星形胶质细胞参与神经元回路的证据并不支持缺乏对这些群体及其结构域中的神经元的详细解剖学描述14。
病毒载体转导程序以及转基因动物（TG）已被推广为研究大脑结构，功能和细胞相互作用的工具集15，16。利用不同的启动子允许根据其遗传特性，激活水平17，18或投射靶标靶向特定细胞。不同的病毒可以在不同的人群中表达不同颜色的荧光基团。病毒可以与TG中荧光团的内源性表达相结合，或者TG动物可以在不需要病毒的情况下使用。这些技术被广泛用于神经元标记，一些实验室已经开始使用它们，并专门用于靶向其他细胞类型（如星形胶质细胞5，9，19）进行修改。
CLARITY技术于2013年20，21年首次描述，通过使整个大脑透明，同时保持微观结构完整，能够研究厚厚的脑切片。通过结合两种方法 - 病毒载体转导和组织清除 - 现在可以选择检查不同细胞类型群体之间的空间相互作用。大多数星形胶质细胞 - 神经元相互作用研究是在薄脑切片上进行的，由于其大结构域导致不完全星形胶质细胞的图像，从而从根本上限制了分析细胞的数量。使用CLARITY技术可以同时对大规模体积的细胞群进行单细胞分辨率表征。对清晰大脑中荧光标记的细胞群进行成像并不能提供突触分辨率，但可以彻底表征星形胶质细胞与各种神经元细胞类型之间的空间相互作用。
出于这个原因，我们利用这些最先进的技术来研究整个背侧CA1星形胶质细胞的特性，对所有层层（层状镭骨，锥体层和地层Oriens）进行成像。我们测量了数以万计的星形胶质细胞（病毒外显率为>96%5），从而分析了CA1周围整个星形细胞群体的信息。通过神经元标志物的有效外显率，我们可以记录整个CA1星形胶质细胞群与四种类型的神经元细胞之间的相互作用 - 小白蛋白（PV），生长抑素（SST），VIP抑制神经元和兴奋性锥体细胞9。
使用来自TG动物的荧光和不同颜色的病毒载体（所有抑制性细胞）的组合进行了几次实验，而其他（兴奋性）利用两种病毒载体在不同启动子下表达不同的荧光团9。本文提出了一个详细的方案，包括标记大脑中所需细胞，使用修改后的CLARITY程序使大脑透明，以及使用各种程序和软件成像和分析完整的大脑结构。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AAV1-GFAP::TdTomato</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect astrocytes</td>
		</tr>
		<tr>
			<td>AAV5-CaMKII::eGFP</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect neurons</td>
		</tr>
		<tr>
			<td>AAV5-CaMKII::H2B-eGFP</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect neuronal nuclei</td>
		</tr>
		<tr>
			<td>AAV5-CaMKII::TdTomato</td>
			<td>ELSC Vector Core Facility (EVCF)</td>
			<td></td>
			<td>viral vector used to detect neurons</td>
		</tr>
		<tr>
			<td>Acrylamide (40%)</td>
			<td>Bio-rad</td>
			<td>#161-0140</td>
			<td></td>
		</tr>
		<tr>
			<td>Bisacrylamide (2%)</td>
			<td>Bio-rad</td>
			<td>#161-0142</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma</td>
			<td>#B7901</td>
			<td>Molecular weight - 61.83 g/mol</td>
		</tr>
		<tr>
			<td>Confocal microscope, scanning, FV1000</td>
			<td>Olympus</td>
			<td></td>
			<td>4x objective (UPlanSApo, 0.16 NA)</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane, UK</td>
			<td></td>
			<td>A software that allows 3D analysis of images</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>#S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 4%</td>
			<td>EMS</td>
			<td>#15710</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear</td>
			<td>SunJin lab</td>
			<td>#RC147002</td>
			<td></td>
		</tr>
		<tr>
			<td>RapiClear CS</td>
			<td>SunJin lab</td>
			<td>#RCCS002</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma</td>
			<td>#L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>SyGlass software</td>
			<td></td>
			<td></td>
			<td>A software that allows 3D analysis of images using virtual reality</td>
		</tr>
		<tr>
			<td>Tris base 1 M</td>
			<td>Bio-rad</td>
			<td>#002009239100</td>
			<td>Molecular weight - 121.14 g/mol</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ChemCruz</td>
			<td>#sc-29112A</td>
			<td></td>
		</tr>
		<tr>
			<td>Two photon microscope</td>
			<td>Neurolabware</td>
			<td></td>
			<td>Ti:sapphire laser (Chameleon Discovery TPC, Coherent), GaAsP photo-multiplier tubes (Hamamatsu, H10770-40) , bandpass filter (Semrock), water immersion 16x objective (Nikon, 0.8 NA)&#160;</td>
		</tr>
		<tr>
			<td>VA-044 Initiator</td>
			<td>Wako</td>
			<td>#011-19365</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of spatial interaction between astrocytes and neurons in cleared brains

使用CLARITY方法将病毒载体转导和组织清除相结合，可以同时研究几种类型的脑细胞及其相互作用。病毒载体转导能够在同一组织内以不同的荧光颜色标记不同的细胞类型。细胞可以通过活动或投射进行遗传识别。使用改进的CLARITY方案，星形胶质细胞和神经元的潜在样本量增加了2-3个数量级。CLARITY的使用允许对完整的星形胶质细胞进行成像，这些星形胶质细胞太大而无法将其全部放入切片中，并检查体细胞及其所有过程。此外，它还提供了研究星形胶质细胞与不同神经元细胞类型之间的空间相互作用的机会，即每个星形细胞域中锥体神经元的数量或星形胶质细胞与特定抑制神经元群体之间的接近程度。本文详细介绍了如何应用这些方法。

成功清除厚脑组织切片会导致一系列新的问题，这些问题可以询问有关大细胞群的特性，而不是单个细胞或相邻细胞群的特性。为了获得成功的结果，应该严格遵守CLARITY协议，因为需要考虑广泛的参数来减少样品之间的差异（例如，透明度百分比，荧光信息，肿胀度参数）。
图1 描述了从荧光蛋白表达验证到清除方法所有阶段的整个清除过程。 <strong cl...

Watch this Scientific Journal Video about Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains at JoVE.com

Investigation of Spatial Interaction Between Astrocytes and Neurons in Cleared Brains

使用CLARITY方法结合病毒载体转导和大脑清除，可以同时研究大量神经元和星形胶质细胞。

研究清除大脑中星形胶质细胞和神经元之间的空间相互作用

Combining viral vector transduction and tissue clearing using the CLARITY method makes it possible to simultaneously investigate several types of brain ...

2013年首次描述的清晰度技术是一种组织清除技术，可以对大量细胞，血管进行采样，因此即使是蛋白质，在厚厚的脑切片中也能以单细胞分辨率。该技术通过使整个大脑透明来研究完整的微观结构。该协议是用于组织清除的简单芯片和直接管道。该技术可用于清除除脑组织外的其他系统中的组织。首先，使用石蜡膜密封管子和盖子，并在盖子上刺穿两个孔。然后使用内径为5毫米的柔性管和末端连接的19号针从罐中输送氮气。通过将管道连接到管上并允许在室温下进行气体交换30分钟来开始脱气。拆下管道，立即用造型粘土密封孔。然后将脱气密封管转移到37摄氏度的浴中3 1/2小时以聚合水凝胶溶液。将大脑从管中取出。使用实验室湿巾，去除大脑周围的聚合水凝胶，确保没有残留的凝胶附着在大脑表面。将大脑分成厚片，其中包含所有感兴趣的区域。将切片放入第一个透明溶液中，并在37摄氏度下孵育，以70 RPM旋转24小时。同时，准备一个穿孔管来放置大脑。将穿孔管放入装有第二个清除溶液的烧杯中搅拌装置上。然后将大脑放入烧杯中，并用铝箔密封以防止漂白。组织变得透明后，将大脑转移到PBST中，在37摄氏度下孵育，以70 RPM旋转24小时。用新鲜的PBST替换溶液，并继续孵育24小时。然后将大脑在室温下转移到PBS中24小时。24小时后，从PBS中取出大脑并将其转移到屈光率匹配溶液中。将大脑在37摄氏度下孵育过夜。首先，将样品放在载玻片的中间。使用热胶，在载玻片的边缘形成壁，几乎与组织一样高。确保在其中一个角落留出一个小间隙。当热胶层接近脑切片的高度时，在样品上滴一到两滴折射率匹配溶液以润湿上表面并防止气泡形成。当热胶仍然是液体时，用盖玻片密封顶部，尽可能均匀地放置，然后用折射率匹配溶液填充腔室。用热胶合上缝合缝隙。如果热胶壁延伸到幻灯片的边界之外，请切割延伸边缘。如果使用油浸物镜进行成像，则在盖玻片上方的壁上再添加两到三毫米的胶水，以保留浸没液。在此方案之后，脑组织将被清除。使用该协议制备了清晰的脑切片，以可视化小鼠海马体CA1区域的星形胶质细胞和神经元群体。所有星形胶质细胞表达tdTomato和兴奋性神经元在其细胞核中表达H2B-GFP。为澄清组织准备的腔室最适合在双光子或共聚焦显微镜下成像。使用双光子显微镜，在小鼠海马体CA1区域的清除部分观察到超过300个星形胶质细胞。红色和锥体细胞中的海马星形胶质细胞，绿色的体细胞是可见的，厚厚的透明组织代表了这两种细胞类型之间的空间接近。使用扫描激光共聚焦显微镜，在整个半球上追踪来自兴奋性神经元的轴突束。绿色的束状物从背侧海马体向超级乳腺体延伸。红色束从乳腺体到它们在发泄海马体的起源。温度、浓度和孵育时间等因素影响了澄清度程序的结果。因此，方案每一步的精确性对于成功实现透明组织至关重要。使用这种技术，我们测量几种大脑结构中神经元和星形胶质细胞之间的距离。该协议允许分析比以前的研究多两到三个数量级的细胞。

Research

JoVE Journal

Neuroscience

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Isolation and Culture of Mouse Cortical Astrocytes

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees (Apis mellifera L.)

Tactile Conditioning And Movement Analysis Of Antennal Sampling Strategies In Honey Bees Apis mellifera L.

触觉空调和取样策略在蜜蜂触角的运动分析（<em&gt;蜜蜂</em&gt; L.）

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability1, 2. The worker ...

An Engulfment Assay: A Protocol to Assess Interactions Between CNS Phagocytes and Neurons

一个吞噬含量：一个协议，以评估之间的中枢神经系统吞噬细胞和神经元的相互作用

Phagocytosis is a process in which a cell engulfs material (entire cell, parts of a cell, debris, etc.) in its surrounding extracellular environment and ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

斑马鱼脑幼虫神经元、巨噬细胞和小胶质的分离和 RNA 提取

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

High-Resolution 3D Imaging of Rabies Virus Infection in Solvent-Cleared Brain Tissue

溶剂清除脑组织狂犬病病毒感染的高分辨率3D成像

The visualization of infection processes in tissues and organs by immunolabeling is a key method in modern infection biology. The ability to observe and ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

Establishment of an Electrophysiological Platform for Modeling ALS with Regionally-Specific Human Pluripotent Stem Cell-Derived Astrocytes and Neurons

建立区域特异性人多能干细胞衍生星形胶质细胞和神经元模拟ALS的电生理平台

Human pluripotent stem cell-derived astrocytes (hiPSC-A) and neurons (hiPSC-N) provide a powerful tool for modeling Amyotrophic Lateral Sclerosis (ALS) ...